This invention relates to a dough composition, i.e., a mixture to be baked, particularly an improved dough composition for bread. More particularly, it relates to incorporation of an effective amount of an enzyme having raw starch decomposing activity in dough as a substitute of a conventional emulsifying agent. A baked product prepared therefrom has improved softness and is slow in getting hard.
In preparing bread, various oxidizing agents, reducing agents, enzymes, emulsifying agents, and the like have been used as bread improvers.
Conventional oxidizing agents as dough conditioners include potassium bromate, potassium iodate, ammonium persulfate, and L-ascorbic acid. Conventional reducing agents include cystine. Further, enzymes (e.g., glucose oxidase, catalase, xcex1-amylase, and hemicellulase), emulsifying agents (e.g., DATA-DATEM, CSL, SSL, and lecithin) have also been used in combination as a bread improver.
The phenomenon of bread""s hardening with time has not been elucidated completely, but it is considered that bread usually increases hardness with aging of starch or as the molecules of starch associate to form a crystalline region. Hardening of bread is a big economical problem to bread manufacturers and consumers. That is, hardening of bread limits not only the shelf life of bread in retail stores but also the period consumers can keep in store. Various emulsifying agents known to be effective in extending the storage life of bread have been used.
However, the increase of consumers"" resistance to the use of chemical additives in foods has accelerated the necessity to reduce or avoid addition of emulsifying agents. Therefore, use of the above-mentioned enzymes as a promising substitute for emulsifying agents has been given studies. Various types of enzymes that have been applied to bread manufacture are used for the improvement of processing and product characteristics in bread manufacture, and some of them have been used for suppression of hardening.
However, the conventional enzymatic dough conditioners are insufficient as a perfect substitute for an emulsifying agent and unsatisfactory with respect to workability and oven spring of dough. It has therefore been keenly demanded to develop a dough conditioner which is naturally occurring and capable of suppressing hardening of baked food.
In order to overcome the above problems, the inventors of the present invention have conducted extensive investigations into a variety of enzymes. As a result, they have found that addition of an effective amount of an enzyme preparation having raw starch decomposing activity to flour not only brings about marked improvement in processing characteristics in bread making but is highly effective in keeping softness of baked products, and thus completed a method for producing baked products using the enzyme.
Thus, the present invention provides a dough composition comprising not less than 15 units of a raw starch decomposing enzyme per kilogram of a raw material to be baked, which may further contain glucose oxidase and/or hemicellulase, a process for production thereof, and a baked food obtained therefrom.
The term xe2x80x9craw material to be bakedxe2x80x9d as used herein means a material containing starch such as plain flour (a powder of wheat, hereinafter simply referred to as flour), powder of other cereal crops such as rye, and mixtures thereof. The term xe2x80x9cdoughxe2x80x9d as used herein means a composition which comprises the raw material to be baked and a liquid for kneading (e.g., water, a dairy component, an egg component, etc.). The dough may also contain sugars, sodium chloride, shortening, yeast, etc. The dough is preferably a mixture ready to be steamed, baked or fried which is prepared by kneading a mix comprising a raw material to be baked such as flour, etc. as an essential material and other materials, such as fats and oils, saccharides (sugar etc.), dairy products (milk etc.), eggs, yeast, etc. and, if desired, various additives such as enzymes and emulsifying agents, together with liquid such as water, milk, and egg. The dough includes not only general one for making bread, either leavened or unleavened, but one for making doughnuts, pies, pizzas, pancakes, sponge cakes, crepes, rice cake, bun, etc. Therefore, the term xe2x80x9cdoughxe2x80x9d is intended to include not only xe2x80x9cdoughxe2x80x9d in its narrow sense of the word but pastry and batter. The term xe2x80x9cbakexe2x80x9d or xe2x80x9cbakingxe2x80x9d as used herein means steaming, baking, frying, etc. in addition to usual baking.
The present invention relates to dough prepared by adding an enzyme having raw starch decomposing activity to a starch, such as flour, as a raw material to be baked in an amount of not less than 15 units per kilogram of the raw material. The starch content of the raw material to be baked such a flour is generally about 70%. The present invention further relates to a method for preparing a baked food characterized by a combined use of the enzyme having raw starch decomposing activity and various oligosaccharide producing enzymes, such as xcex1-amylase, xcex2-amylase, glucoamylase, cyclodextrin glucanotransferase, hemicellulase, xylanase, pentosanase, xcex2-glucanase, cellulose, mannase, arabinofuranosidase, debranching enzymes, maltotriose producing enzymes, maltotetraose producing enzymes, maltopentaose producing enzymes, and maltohexaose producing enzymes, glucose oxidase, catalase, sulfhydryl oxidase, lipoxygenase, peroxidase, protease, peptidase, glutaminase, transglutaminase, lipase, phosphatase, etc. A combined use with glucose oxidase and/or hemicellulase is particularly preferred.
The baked products made from the dough of the present invention keep softness for an extended period of time. Where other enzymes are used in combination, the dough exhibits improved elasticity and assumes a tacky-dry state on its surface which greatly contributes to workability. The bread or the like prepared from the dough of the present invention also exhibits satisfactory properties.
With respect to starch in the raw state, general amylase species can hardly decompose the long-chain xcex1-1xe2x86x924 glucan. The terminology xe2x80x9cenzyme having raw starch decomposing activityxe2x80x9d or xe2x80x9craw starch decomposing enzymexe2x80x9d as used herein is intended to mean an amylase which can act on raw starch, i.e., starch before gelatinization. Known amylases of this type include those produced by microorganisms belong to genera Aspergillus, Bacillus, Rhizopus, and Zoogloea. More specifically, Gluczyme AF6 (a glucoamylase preparation of Rhizopus niveus origin, available from Amano Pharmaceutical Co., Ltd.), Glucoamylase Amano (a glucoamylase preparation of Aspergillus niger origin, available from Amano Pharmaceutical Co., Ltd.), and a raw starch decomposing enzyme of Zoogloea ramigera KO-159 origin (see Unexamined Published Japanese Patent Application 4-141082) can be mentioned as examples.
The raw starch decomposing enzyme according to the present invention is not particularly limited as long as it shows raw starch decomposing activity. The ratio of the raw starch decomposing activity per the soluble starch decomposing activing (%) as measured in the methods described below is preferably 0.1% or more, more preferably 1% or more, and most preferably 3% or more.
The raw starch decomposing enzyme introduced into dough hydrolyses raw starch constituting the raw material, such as flour. Since amylases that have been used as a bread improver hardly act on un-gelatinized starch, they are ineffective in dough in which starch is in a raw state. To the contrary, the enzyme having raw starch decomposing activity as used in the present invention acts on the raw starch from the stage of dough preparation to produce glucose, etc. thereby to accelerate fermentation by yeast.
The raw starch decomposing activity is determined as follows.
Measurement of Raw Starch Decomposing Activity
In a 100 ml-volume Erlenmeyer flask is put 10 ml of a 50 mg/ml suspension of fluor in a 0.05M acetate buffer (pH 5.0), and 0.1 ml of an enzyme solution is added thereto, followed by reacting for 1 hour while shaking in a thermostat kept at 40xc2x0 C. at a frequency of 120/min with an amplitude of 3 cm so that the substrate may not stand still on the bottom of the flask. After the reaction, the reaction mixture is centrifuged at 3,000 rpm for 10 minutes to obtain a supernatant liquor. The produced reducing sugar in the supernatant liquor is quantitatively determined with a 3,5-dinitrosalicylic acid reagent based on a calibration curve of glucose. Enzyme activity which produces reducing sugar corresponding to 1 mg of glucose per 1 minute under the above conditions is taken as one unit.
Measurement of Soluble Starch Decomposing Activity
In a test tube is put 1 ml of a 10 mg/ml soluble starch solution in a 0.075 mol/l acetic acidxe2x80xa2sodium acetate buffer (pH 5.0), and 0.5 ml of an enzyme solution is added thereto, followed by reacting for 15 minutes at 40xc2x0 C. After the reaction, 3 ml of 3,5-dinitrosalicylic acid reagent (containing 10 g NaOH, 10 g of 3,5-dinitrosalicylic acid, 0.5 g of anhydrous sodium sulfate, and 40 ml of 5% phenol per 1 l of reagent) was added, and the mixture was heated on a boiling water bath for 20 minutes, followed by cooling with running water. Thereafter, 15.5 ml of water was added and the mixture was subjected to measurement of absorbance at the wavelength of 560 nm. Enzyme activity which produces reducing sugar corresponding to 1 xcexcmol of glucose per 1 minute under the above conditions is taken as one unit.
The raw starch decomposing activity required in the present invention is 15 units or more per kilogram of the raw material to be baked. Less than 15 units cannot manifest the desired effects in improving softness and retarding hardening. A preferred range of the amount of the enzyme to be used is usually from 50 to 700 units.
While the optimum amount of the raw starch decomposing enzyme to be added is subject to variation depending on the kind of the grain as a raw material and the processes involved, it can easily be decided through a baking test.
As previously stated, the raw starch decomposing enzyme can be used in combination with other conventionally employed enzymes of various kinds. The feature of the present invention also lies in that a combined use of other enzymes surprisingly enhances the effects improving workability of dough and bread properties. For example, it has been ascertained that a combination with a general amylase which acts on gelatinized starch, hemicellulase or glucose oxidase produces complementary and synergistic effects.
The amylase which acts on gelatinized starch as referred to above means a commonly used amylase, such as xcex1-amylase, xcex2-amylase or Aspergillus origin or malt origin, and isoamylase.
The hemicellulase includes species of Aspergillus niger origin and of Aspergillus awamori origin, which are known useful for baked products. Hemicellulase Amano 90 (available from Amano Pharmaceutical Co., Ltd.) can be mentioned as a specific example.
The glucose oxidase includes species of Aspergillus niger origin, such as Hyderase 15 (available from Amano Pharmaceutical Co., Ltd.).
Hemicellulase, when added to dough, produces excellent effects in improving such properties as volume and softness of baked products but tends to make the dough too soft and sticky and difficult to handle. These disadvantages of hemicellulase can be eliminated by using glucose oxidase in combination. Besides, a combined use of hemicellulase and glucose oxidase further improves the bread making properties compared with the use of hemicellulase alone.
In the present invention, it is preferable to added 0 to 50,000 units of hemicellulase and/or 0 to 1000 units of glucose oxidase per kilogram of a starch, such as flour. Preferably, these enzymes are used in 5 to 200 ppm, more preferably 20 to 80 ppm.
Although the present invention is not to exclude use of conventional dough conditioners, the use of a raw starch decomposing enzyme according to the present invention makes it possible to produce baked products which keep softness for an extended period of time without using conventional chemical additives such as emulsifying agents.
The activities of amylase that acts on gelatinized starch, hemicellulase, and glucose oxidase can be determined as follows.
Measurement of Amylase Activity
In a 100 ml-volume Erlenmeyer flask is put 10 ml of a substrate. After the flask is allowed to stand in a thermostat set at 40xc2x10.1xc2x0 C. for 10 minutes, 1 ml of an enzyme solution in a 0.05M acetic acid-sodium acetate buffer solution (pH 5.0) is added thereto, followed by shaking well. Immediately thereafter, the flask is placed in a thermostat kept at 40xc2x10.1xc2x0 C. and allowed to stand accurately for 30 minutes. In the flask is put 4 ml of a Fehling""s solution, followed by shaking well. The mixture is placed on a glass ceramic plate set on a gas burner and heated while adjusting the heating power so that the mixture may boil in 60xc2x130 seconds and boiled accurately for 2 minutes. After cooling with running water, 2 ml of a 30% aqueous potassium iodide solution and 2 ml of a 25% aqueous sulfuric acid solution are added to the reaction mixture in this order, immediately followed by titration with a 0.05M aqueous sodium thiosulfate solution (for quantitative determination). The time when the mixture turned white is taken as an end point. Where the end point is difficult to detect, two or three drops of a starch reagent solution are added to the reaction mixture, and titration is carried out until the system turned white. Separately, a blank test is carried out using water in place of the enzyme solution. Enzyme activity which brings about an increase in reducing power corresponding to 10 mg of glucose in 30 minutes is taken as one unit.
The substrate to be used in the above measurement is prepared by suspending 1 g of dried soluble starch in a small amount of water, adding the suspension to about 100 ml of boiling water, boiling the mixture for 5 minutes from the start of boiling, cooling the container with running water, adding 10 ml of a 1M acetic acid-sodium acetate buffer solution (pH 5.0), and adding water to make 200 ml.
Measurement of Hemicellulase Activity
To 1 ml of a substrate solution (10 mg/ml of xylan) is added 3 ml of a 0.1N acetate buffer (pH 4.5), followed by stirring. The solution is preheated at 40xc2x0 C. for 10 minutes. Then 1 ml of an enzyme solution is added thereto, and the system is allowed to react at 40xc2x0 C. for 30 minutes. After the reaction, 2 ml of a Somogyi""s solution is added, followed by shaking, and the reaction mixture is heated in a boiling water bath for 20 minutes. After cooling, 1 ml of an ammonium arsenomolybdate solution is added to the reaction mixture, followed by shaking, and water is added thereto to make 25 ml. The mixture is centrifuged at 3000 rpm for 10 minutes, and the absorbance of the supernatant liquor at a wavelength of 500 nm is measured. Enzyme activity which produces reducing sugar corresponding to 1 mg of xylose per 1 minute is taken as 100 units.
Measurement of Glucose Oxidase Activity
In a quartz cell having a cell thickness of 10 mm are put 2 ml of a phosphate buffer containing 50 mg/ml of phenol, 0.5 ml of a substrate solution (2.5 g/ml of glucose), a peroxidase solution (0.5 ml; 25 units/ml of peroxidase), and a 4-aminoantipyrine solution (0.1 ml; 4 mg/ml) and allowed to stand at 37xc2x0 C. for 10 minutes. To the mixture is added 0.1 ml of an enzyme solution, stirred well, and allowed to stand at 37xc2x0 C. After 2 minutes"" and 5 minutes"" reacting, the absorbance of the reaction mixture at a wavelength of 500 nm is measured. Separately, a blank test is carried out using water in place of the enzyme solution. Enzyme activity that oxidizes 1 xcexcmol of glucose per 1 minute is taken as one unit.
The enzymes which can be used in the present invention, such as the enzyme having raw starch decomposing enzyme, amylase acting on gelatinized starch, hemicellulase, glucose oxidase, etc. do not necessarily need to be purified preparations. Crude enzyme preparations can be used as well. The enzymes can be obtained by fermentation of microorganisms or a culture of the microorganisms may be used. If desired, the culture can be concentrated or dried.
The dough according to the present invention can further contain various components other than the above-described enzymes known in the art, for example, sugar, edible salt, lecithin, gluten, soybean powder, malt, and other enzymes.
The dough of the present invention can be baked in a usual manner into baked products by any of known bread making methods, such as a straight dough method, a no-time straight dough method, an overnight sponge and dough method, a sponge and dough method, a frozen dough method, and the like.
The present invention achieves considerable retardation of hardening of baked products as well as improvements in volume and softness of baked products. The present invention also brings about improvements in dough properties in terms of handling properties, elasticity, and extensibility in rolling and provides baked products with excellent appearance and palatability.